WO1997024457A1 - Procede et kit pour la detection d'interactions entre plusieurs proteines - Google Patents

Procede et kit pour la detection d'interactions entre plusieurs proteines Download PDF

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WO1997024457A1
WO1997024457A1 PCT/IB1996/001495 IB9601495W WO9724457A1 WO 1997024457 A1 WO1997024457 A1 WO 1997024457A1 IB 9601495 W IB9601495 W IB 9601495W WO 9724457 A1 WO9724457 A1 WO 9724457A1
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protein
gene
dna
host cell
hybrid
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PCT/IB1996/001495
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WO1997024457B1 (fr
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Susan Lautar
Jie Zhang
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Guilford Pharmaceuticals Inc.
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Priority to AU12063/97A priority Critical patent/AU1206397A/en
Publication of WO1997024457A1 publication Critical patent/WO1997024457A1/fr
Publication of WO1997024457B1 publication Critical patent/WO1997024457B1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to a method for detecting the interaction of three or more proteins in an in vivo system through the use of fused genes encoding hybrid proteins.
  • Proteins are complex macromolecules made up of covalently linked chains of amino acids. Each protein assumes a unique three dimensional shape determined principally by its sequence of amino acids. Many proteins consist of smaller units termed domains, which are continuous stretches of amino able to fold independently from the rest of the protein. Some of the important forms of proteins are as enzymes, polypeptide hormones, nutrient transporters, structural components of the cell, hemoglobins, anti-bodies, nucleoproteins, and components of viruses.
  • Multiple protein interactions require three or more proteins to associate.
  • a large number of non- covalent bonds form between the proteins when three or more protein surfaces are precisely matched, and these bonds account for the specificity of recognition.
  • Multiple-protein interactions are involved, for example, in the assembly of enzyme subunits; in antigen-antibody reactions; in forming the supramolecular structures of ribosomes, filaments, and viruses; in transport; and in the interaction of receptors on a cell with growth factors and hormones.
  • Products of oncogenes can give rise to the neoplastic transformation through multiple-protein interactions.
  • some oncogenes encode protein kinases whose enzymatic activity on cellular target proteins leads to the cancerous state.
  • Another example of a protein-protein interaction occurs when a virus infects a cell by recognizing a polypeptide receptor on the surface, and this interaction has been used to design antiviral agents.
  • Protein-protein interactions have been generally studied in the past using biochemical techniques such as cross-linking, co-immunoprecipitation and co- fractionation by chromatography.
  • a disadvantage of these techniques is that interacting proteins often exist in very low abundance and are, therefore, difficult to detect.
  • Another major disadvantage is that these biochemical techniques involve only the proteins, not the genes encoding them. When an interaction is detected using biochemical methods, the newly identified protein often must be painstakingly isolated and then sequenced to enable the gene encoding it to be obtained. Another disadvantage is that these methods do not immediately provide information about which domains of the interacting proteins are involved in the interaction. Another disadvantage is that small changes in the composition of the interacting proteins cannot be tested easily for their effect on the interaction.
  • transcription can be activated through the use of two functional domains of a transcription factor: a domain that recognizes and binds to a specific site on the DNA and a domain that is necessary for activation, as reported by Keegan, et al., Science, 231, 699-704 (1986) and Ma and Ptashne, Cell, 48, 847-853 (1987) .
  • the transeriptional activation domain is thought to function by contracting other proteins involved in transcription.
  • the DNA-binding domain appears to function to position the transeriptional activation domain on the target gene which is to be transcribed. In a few cases now known, these two functions (DNA-binding and activation) reside on separate proteins.
  • UAS G upstream activation site, G indicates the galactose genes
  • C-terminal domain containing acidic regions which is necessary to activate transcription, see Keegan et al . (1986) , supra, and Ma and Ptashne. (1987), supra.
  • the N- terminal domain binds to DNA in a sequence-specific manner but fails to activate transcription.
  • the C- terminal domain cannot activate transcription because it fails to localize to the UAS G see for example, Brent and Ptashne, Cell, 43, 729-736 (1985) .
  • yeast two-hybrid systems of Fields et al . , Nature 340, 245-246 (1989) , also disclosed in U.S. Pat. No. 5,283,173 by Fields et al .
  • the yeast two-hybrid system detects binary (X/Y) interactions between proteins through functional reconstitution of transcription factor GAL4 by associating two fusion proteins, GAL4-DNA binding domain, (BD) -X, and GAL -activation domain, (AD) -Y.
  • the yeast two-hybrid system offers a sensitive genetic selection method to detect and clone physically interactive proteins.
  • Fields et al . Nature 340, 245-246 (1989) ; Fields et al . , Proc. Natl. Acad. Sci. 88, 9578-9582.
  • the two-hybrid system is limited so far as to detect protein interactions involving two components only.
  • the two-hybrid system cannot detect protein (Z)- mediated interactions between proteins (X/Y) where the proteins indirectly interact, e.g. X does not contact Y.
  • the two-hybrid system also cannot detect protein interactions which require modification of the X or Y protein by the Z protein to interact, or have complex confor ational requirements for interaction. None of the aforementioned articles suggests a genetic system to detect three or more protein interactions in vivo using transeriptional activation as an assay.
  • a genetic system that is capable of rapidly detecting which of multiple proteins interact with a known protein, determining which of multiple domains of the proteins interact, and providing the genes for the newly identified interacting proteins has not been available prior to the present invention.
  • RNA molecules are synthesized using a DNA template. Transcription is regulated by specific sequences in the DNA which indicate when and where RNA synthesis should begin. These sequences correspond to binding sites for proteins, designated transcription factors, which interact with the enzymatic machinery used for the RNA polymerization reaction.
  • the present invention provides a method and a kit for detecting interactions between three or more proteins, in vivo, using reconstitution of the activity of a transeriptional activator.
  • This reconstitution makes use of the chimeric genes which express hybrid proteins.
  • three types of hybrid proteins are prepared.
  • the first hybrid contains the DNA-binding domain of a transeriptional activator fused to the first test protein.
  • the second hybrid contains a transeriptional activation domain fused to a second test protein.
  • the third hybrid contains a nuclear localization peptide fused to a third test protein. If the three test proteins are able to interact, they bring into close proximity the two domains of the transeriptional activator. This proximity is sufficient to cause transcription, which can be detected by the activity of a marker gene that contains a binding site for the DNA-binding domain.
  • One advantage of this method is that a multiplicity of proteins can be quickly and simultaneously tested to determine whether any interact with proteins known to be within a protein mediated signal transduction pathway.
  • a DNA library may be screened and DNA fragments encoding such mediator proteins can yield a positive result.
  • a DNA fragment encoding the DNA- binding domain is fused to a DNA fragment encoding the known protein in order to provide one hybrid.
  • a second DNA fragment, encoding the transeriptional activation domain is fused to a DNA fragment encoding a second protein and provides the second hybrid which indirectly interacts with the first hybrid.
  • a library of plasmids can be constructed which may include, for example, total mammalian complementary DNA (cDNA) to encode the third mediating protein.
  • This library is introduced into the cells carrying the first and second hybrids. If any individual plasmid from the library encodes a protein that is capable of interacting with the known proteins, a positive signal may be obtained.
  • the EGFR/Grb2/Sos system described herein can be easily adapted to screen a pDela plasmid cDNA library to look for Grb2- like adaptor proteins.
  • the gene for the newly identified protein is readily available rendering sequencing of the newly identified protein unnecessary.
  • the system can be used to screen for drugs that disrupt the three hybrid complex and turn off ⁇ - galactosidase production within the host cell.
  • drugs may either turn off, down-regulate, or up- regulate signal transduction pathways, e.g. intervening in the tyrosine receptor signal transduction pathway that has been implied in certain pathological conditions such as adenocarcinoma.
  • drugs regulating complex pathways such as protein synthesis, protein modification, protein catabolism, cellular regulation, and the like, can be detected.
  • the system can be of value in the identification of new genes.
  • receptors on the cell surface may be identified for known growth factors, toxins, or surface antigens. Proteins that interact with oncogene-encoded products may be discovered, and these proteins can be of therapeutic value.
  • the system can be used in the design of peptide inhibitors. For example, peptides that interact with enzymes such as proteases or kinases can be identified and then tested in other systems for their ability to inhibit the enzymatic reaction. Peptides that bind to bacterial or viral proteins can be identified and then tested in other systems for their ability to inhibit these bacteria or viruses.
  • the system can be used to test affinity reagents for protein purification. Peptides or protein domains can be identified that interact with the known protein of interest and these may then be used in a purification protocol for the known protein.
  • a broad embodiment of the present invention encompasses a method for detecting multiple protein interactions, the method comprising:
  • a first test protein or fragment thereof that is to be tested for interaction with a second test protein or fragment thereof and at least one mediating test protein or fragment thereof;
  • a second chimeric gene that is capable of being expressed in the host cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising:
  • test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof and with at least one mediating test protein or fragment thereof;
  • each mediating chimeric gene comprising a DNA sequence that encodes a mediating hybrid protein, the mediating hybrid protein comprising:
  • a nuclear localization peptide (i) a nuclear localization peptide; and (ii) a mediating test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof and the second test protein or fragment thereof ; wherein interaction between the first test protein, the second test protein, and the mediating test protein in the host cell causes the transeriptional activation domain to activate transcription of the detectable gene; (e) introducing the first chimeric gene, the second chimeric gene, and the mediating chimeric gene into the host cell;
  • An alternate embodiment of the present invention provides a method for detecting an interaction between a first test protein, a second test protein, and a third test protein, the method comprising:
  • test protein or fragment thereof that is to be tested for interaction with a second test protein or fragment thereof and at least one third test protein or fragment thereof;
  • the third chimeric gene comprising a DNA sequence that encodes a third hybrid protein, the third hybrid protein comprising: (i) a nuclear localization peptide; and (ii) a third test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof and the second test protein or fragment thereof; wherein interaction between the first test protein, the second test protein, and the third test protein in the host cell causes the transeriptional activation domain to activate transcription of the detectable gene;
  • a preferred embodiment of the present invention provides a method for detecting an interaction between a first test protein, a second test protein, and a third test protein, the method comprising: (a) providing a Saccharomyces cerevisiae host cell containing a GALl-lacZ gene wherein the GALl-lacZ gene expresses a /3-galactosidase protein when the GALl-lacZ gene is activated by an amino acid sequence including a transeriptional activation domain of yeast transcription factor GAL4 when the transeriptional activation domain is in sufficient proximity to the GALl-lacZ gene, and containing a GAL1-His3 gene, when the GAL1-His3 gene is activated providing histidine-independent yeast growth;
  • test protein or fragment thereof that is to be tested for interaction with a second test protein or fragment thereof and at least one third test protein or fragment thereof;
  • the transeriptional activation domain and (ii) a second test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof and the third test protein or fragment thereof; (d) providing a third DNA plasmid, the third DNA plasmid comprising a third chimeric gene that is capable of being expressed in the nuclei of the host cell, the third chimeric gene comprising a DNA sequence that encodes a third hybrid protein, the third hybrid protein comprising:
  • an SV40 T antigen nuclear localization peptide sequence (i) an SV40 T antigen nuclear localization peptide sequence; and (ii) a third test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof and the second test protein or fragment thereof; wherein interaction between the first test protein, the second test protein, and the third test protein in the host cell causes the transeriptional activation domain to activate transcription of the GAL-lacZ gene; (e) introducing the first DNA plasmid, the second DNA plasmid, and the third DNA plasmid into the host cell;
  • a kit for detecting interaction between a first test protein and a second test protein where the proteins interact through mediation by one or more third proteins, the kit comprising:
  • a container (a) a container; (b) a host cell is provided within the container, the host cell contains a detectable gene having a binding site for a DNA-binding domain of a first hybrid protein, the binding site positioned so that the detectable gene expresses a detectable protein when the detectable gene is activated by a transeriptional activation domain encoded by a second vector, activation of the detectable gene occurs when the transeriptional activation domain is in sufficient proximity to the detectable gene, the host cell, by itself, incapable of expressing a protein having a function of a first marker gene, a second marker gene, the DNA- binding domain, or the transeriptional activation domain; (c) a first vector is provided within the container, the first vector contains a promoter and a transcription termination signal functionally associated with a first chimeric gene in order to direct the transcription of the first chimeric gene, the first chimeric gene includes a DNA sequence that encodes a DNA-binding domain and at least one unique restriction site
  • a third vector is provided within the container, the third vector contains a third chimeric gene, the third chimeric gene includes a promoter and a transcription termination signal to direct transcription, the third chimeric gene includes at least one unique restriction site to insert a DNA sequence encoding a third test protein or protein fragment into the vector in such a manner that the third test protein is capable of being expressed as part of a hybrid protein, the third vector also includes a means for self-replicating in the host cell, a nuclear localization peptide, and a third marker gene, the expression of which in the host cell permits selection of cells containing the third marker gene from cells that do not contain the third marker gene.
  • FIG.l is a representational map of the plasmid pDela for expression of a third protein-hybrid in yeast nuclei .
  • FIG.2 is a schematic representation of three DNA fragments encoding the ternary complex of EGF receptor, Grb2 and Sos in the yeast three-hybrid system.
  • FIGS.3A is a schematic representations of the three-hybrid system where interaction of proteins X and Y requires mediation by protein Z to reconstitute transeriptional activation of GAL4 activity.
  • FIG.3B is a schematic representation of the three-hybrid system where protein Y only binds to a new composite contour created by the combination of protein X and protein Z to achieve reconstitution of GAL4 transeriptional activation.
  • FIG . 3C is a schematic representation of the three -hybrid system where Y binds to X only after X is modified by Z to achieve reconstitution of GAL4 transeriptional activation.
  • a method for detecting the interaction between three or more test proteins includes providing a host cell, preferably a yeast cell, most preferably Saccharomyces cerevisiae or Schizosaccharomyces pombe.
  • the host cell contains a detectable gene having a binding site for the DNA- binding domain of the transeriptional activator, such that the detectable gene expresses a detectable protein when the detectable gene is transeriptionally activated. Such activation occurs when the transeriptional activation domain of a transeriptional activator is brought into sufficient proximity to the DNA-binding domain of the transeriptional activator.
  • a first chimeric gene is provided which is capable of being expressed in the host cell.
  • the first chimeric gene may be present in a chromosome of the host cell.
  • the first chimeric gene comprises a DNA sequence that encodes a first hybrid protein.
  • the first hybrid protein contains a DNA-binding domain that recognizes the binding site on the detectable gene in the host cell.
  • the first hybrid protein contains a first test protein or protein fragment which is to be tested for interaction with a second test protein or protein fragment and a third test protein or protein fragment.
  • the first hybrid is carried on the pGBT9 plasmid.
  • a second chimeric gene is provided which is capable of being expressed in the host cell.
  • the first, second and third chimeric genes are introduced into the host cell in the form of plasmids.
  • one chimeric gene is present in a chromosome of the host cell and the other chimeric genes are introduced into the host cell as part of a plasmid or plasmids.
  • the second hybrid is on the pGAD424 plasmid.
  • the second chimeric gene contains a DNA sequence that encodes a second hybrid protein.
  • the second hybrid protein contains a transeriptional activation domain.
  • the second hybrid protein also contains a second test protein or a protein fragment which is to be tested for interaction with the first and third test proteins or protein fragments.
  • the DNA-binding domain of the first hybrid protein and the transeriptional activation domain of the second hybrid protein are ' derived from transeriptional activators having separate DNA-binding and transeriptional activation domains.
  • These separate DNA-binding and transeriptional activation domains are also known to be found in the yeast GAL4 protein, and are also known to be found in the yeast GCN4 and ADR1 proteins . Many other proteins involved in transcription also have separable binding and transeriptional activation domains which make them useful for the present invention.
  • the DNA-binding domain and the transeriptional activation domain may be from different transeriptional activators.
  • the third chimeric gene contains a DNA sequence that encodes a third hybrid protein or protein fragment.
  • the DNA sequence is constructed on a plasmid (pDela) which is compatible with two widely used plasmids, e.g. pGBT9 and pGAD424. Most of the currently used two-hybrid plasmids, e. g. pGBT9 and pGAD424, employ Leu + and Trp + genes as selection markers for maintaining them in Leu " Trp " yeast strains.
  • the plasmid encoding the third hybrid protein also contains a selection marker gene, e.g. Ura3 + , so that triple transformants of the hybrid plasmids, e.g.
  • pDela, pGBT9 and pGAD424 can be selected on an appropriate yeast background, e.g. Ura ' Trp ' Leu " .
  • High level constitutive transcription of the third hybrid is driven by an appropriate strong promoter, such as that of house-keeping alcohol dehydrogenase gene 1 (PADH1) .
  • PADH1 house-keeping alcohol dehydrogenase gene 1
  • the third hybrid protein is targeted to yeast nuclei by a nuclear localization signal, e.g. the SV40 T antigen NLS sequence.
  • the third hybrid protein may also be encoded on a library of plasmids that contain genomic, cDNA or synthetically generated DNA sequences fused to the DNA sequence encoding the nuclear localization sequence.
  • the interaction between the three test proteins in the host cell therefore, causes the transeriptional activation domain to activate transcription of the detectable gene.
  • the method is carried out by introducing the three chimeric genes into the host cell.
  • the host cell is subjected to conditions under which the hybrid proteins are expressed in sufficient quantity for the detectable gene to be activated.
  • the cells are then tested for their expression of the detectable gene to be a greater degree than in the absence of an interaction between the three test proteins.
  • the test proteins may be derived from bacterial proteins, viral proteins, oncogene-encoded proteins, growth factors or an enzymes.
  • the third test protein may be derived from a library of plasmids as described above.
  • the method of the present invention, as described above, may be practiced using a kit for detecting interaction between a first test protein and a second test protein where the proteins interact through mediation by one or more "third" proteins.
  • the kit includes a container, three vectors, and a host cell.
  • the first vector contains a promoter and may include a transcription termination signal functionally associated with the first chimeric gene in order to direct the transcription of the first chimeric gene.
  • the first chimeric gene includes a DNA sequence that encodes a DNA-binding domain and a unique restriction site(s) for inserting a DNA sequence encoding a first test protein or protein fragment in such a manner that the first test protein is expressed as part of a hybrid protein with the DNA-binding domain.
  • the first vector also includes a means for replicating itself in the host cell and in bacteria. Also included on the first vector is a first marker gene, the expression of which in the host cell permits selection of cells containing the first marker gene from cells that do not contain the first marker gene.
  • the first vector is plasmid such as pGBT9.
  • the kit also includes a second vector which contains a second chimeric gene.
  • the second chimeric gene also includes a promoter and a transcription termination signal to direct transcription.
  • the second chimeric gene also includes a DNA sequence that encodes a transeriptional activation domain and a unique restriction site(s) to insert a DNA sequence encoding the second test protein or protein fragment into the vector, in such a manner that the second test protein is capable of being expressed as part of a hybrid protein with the transeriptional activation domain.
  • the DNA-binding domain of the first hybrid protein and the transeriptional activation domain of the second hybrid protein are derived from transeriptional activators having separate DNA-binding and transeriptional activation domains.
  • the second vector further includes a means for replicating itself in the host cell and in bacteria.
  • the second vector also includes a second marker gene, the expression of which in the host cell permits selection of cells containing the second marker gene from cells that do not contain the second marker gene.
  • the kit also provides a third vector, which contains a third chimeric gene.
  • the third chimeric gene also includes a promoter and a transcription termination signal to direct transcription.
  • the third chimeric gene includes a unique restriction site(s) to insert a DNA sequence encoding the third test protein or protein fragment into the vector in such a manner that the third test protein is capable of being expressed as part of a hybrid protein.
  • the third hybrid protein may be encoded on a library of plasmids DNA sequences fused to the DNA sequence encoding the nuclear localization sequence.
  • the third vector also includes a means for replicating itself in the host cell and in bacteria.
  • the third vector includes a nuclear localization peptide, such as SV40 T antigen nuclear localization sequence, and a second marker gene, the expression of which in the host cell permits selection of cells containing the third marker gene from cells that do not contain the third marker gene.
  • the kit includes a host cell, preferably a yeast strain of Saccharomyces cerevisiae or Schizosac ⁇ charomyces pombe.
  • the host cell contains the detectable gene having a binding site for the DNA- binding domain of the first hybrid protein.
  • the binding site is positioned so that the detectable gene expresses a detectable protein when the detectable gene is activated by the transeriptional activation domain encoded by the second vector. Activation of the detectable gene is possible when the transeriptional activation domain is in sufficient proximity to the detectable gene.
  • the host cell by itself, is incapable of expressing a protein having a function of the first marker gene, the second marker gene, the third marker gene, the DNA-binding domain, or the transeriptional activation domain.
  • the detectable gene may encode an enzyme or other product that can be readily measured. Such measurable activity may include the ability of the cell to grow only when the marker gene is transcribed, or the presence of detectable enzyme activity only when the marker gene is transcribed.
  • Various other markers are well known within the skill of workers in the art.
  • the cells containing the three hybrid proteins are incubated in a appropriate medium and the culture is monitored for the measurable activity.
  • a positive test for this activity is an indication that the test proteins have interacted.
  • Such interaction between the multiple proteins brings their respective DNA- binding and transeriptional activation domains into sufficiently close proximity to cause transcription of the marker gene.
  • the basic strategy of the testing method includes preparing the system of three hybrid proteins containing domains of a yeast transeriptional activator, as described.
  • the first hybrid contains the DNA-binding domain of a transeriptional activator fused to the first test protein.
  • the second hybrid protein contains a transeriptional activation domain fused to the second test protein.
  • the third hybrid protein mediates assembly of the three-protein complex involving the three hybrids. If the three test proteins are able to interact, they bring into close proximity the two domains of the transeriptional activator. This proximity is sufficient to cause transcription, which can be detected by the activity of a marker gene that contains a binding site for the DNA-binding domain.
  • a yeast strain e.g.
  • BY1361 is used that carries several genes under the regulation of UAS G and therefore to bind the GAL4 DNA-binding domain.
  • GALl-lacZ which contains the E. coli lacZ gene encoding ⁇ - galactosidase.
  • X-gal is an abbreviation for 5- bromo-4-chloro-3-indolyl-j ⁇ -D-galactopyranoside.
  • X- gal is cleaved by .-galactosidase, producing a color change. Therefore ⁇ -galactosidase activity, detected by liquid assay or by colony color on appropriate media, is a measure of GAL4 function.
  • FIG. 3A schematically illustrates GAL4- hybrid complex 10 showing the binding of the GAL4- first hybrid fusion protein 12 having a DNA-binding domain 14.
  • the GAL4-second hybrid fusion protein 16 is shown having a transeriptional activation domain 18.
  • the third hybrid protein 20 is shown mediating the protein interaction which supplies the proper placement of the binding domain and the transeriptional activation domain.
  • the GAL4-hybrid complex 10, containing both domains 14 and 18, provides potent activation of transcription of the GALl-lacZ gene 22 when yeast are grown on galactose media. Transcription of the GALl-lacZ gene 22 is indicated by the arrow 24.
  • the system is dependent on a number of conditions to properly carry out the method of this invention.
  • the first interacting protein X must not, itself, carry as activation domain for the marker. Otherwise the activation domain would allow transcription of the marker gene as soon as the vector encoding only the GAL4 DNA-binding domain fused to the first interacting protein X is introduced.
  • the interaction between the first test protein X and the second or third test protein Y, protein Z must be capable of occurring within the yeast nucleus.
  • the GAL4 activation domain portion of the hybrid containing the second test protein Y must be accessible to the transcription machinery of the cell to allow transcription of the marker gene. Should any of these conditions not exist, the system may be modified for use by constructing hybrids that carry only portions of the interacting proteins X Y, and Z, and thus meet these conditions.
  • This system can be used to genetically select for proteins that mediate interactions of known proteins, provided the gene encoding the known proteins are available.
  • Yeast containing the known proteins as a hybrid with the GAL4 DNA activation domain and GAL4 DNA-binding domain can be transformed with a clone bank of genomic or cDNA sequences fused to the nuclear localization domain.
  • the triple transformants can be selected for their ability to grow on histidine drop ⁇ out media, or screened for blue color on indicator plates for those transformants able to express the GALl-lacZ fusion.
  • the reporter gene function can be served by any of a large variety of genes, such as genes encoding drug resistance or metabolic enzymes.
  • the function of GAL4 can be served by any transeriptional activator that has separable domains for DNA-binding and for transeriptional activation. Indeed, any protein, even one that is not a transeriptional activator, that has two separable functions can be used to establish a similar genetic system to detect multiple-protein interactions.
  • the method of the present invention can be applied more generally to any detectable function requiring separable domains of an amino acid sequence which can be reconstituted.
  • This general embodiment of the present invention detects interaction between three test proteins.
  • the method includes providing a host cell which is defective in a detectable function requiring three protein interaction.
  • the detectable function is provided by an amino acid sequence having separable domains.
  • the amino acid sequence includes a first domain and a second domain which are capable of producing the detectable function when they are brought together by a third hybrid protein in sufficient proximity to each other in the host cell.
  • chimeric genes are provided that are capable of being expressed in the host cell .
  • the first chimeric gene includes a DNA sequence that encodes a first hybrid protein.
  • the first hybrid protein contains the first domain of the amino acid sequence.
  • the first hybrid protein also contain a first test protein or protein fragment which is to be tested for interaction with a second test protein or protein fragment and a third test protein or protein fragment.
  • a second chimeric gene is provided which is capable of being expressed in the host cell .
  • the second chimeric gene contains a DNA sequence that encodes a second hybrid protein.
  • the second hybrid protein contains the second domain of the amino acid sequence.
  • the second hybrid protein also contains a second test protein or protein fragment which is to be tested for interaction with the first and third test proteins or protein fragments.
  • a third chimeric gene includes a DNA sequence that encodes a third hybrid protein.
  • the third hybrid protein contains a third test protein or protein fragment which is to be tested for interaction with the first and second proteins or protein fragments.
  • the interaction between the test proteins in the host cell causes the function of the amino acid sequence to be reconstituted.
  • the method is thus carried out by introducing the chimeric genes into the host cell.
  • the host cell is subjected to conditions under which the hybrid proteins are expressed in sufficient quantity for the function of the amino acid sequence to be reconstituted.
  • the cells are then tested to determine whether their expression of the function of the amino acid sequence has been reconstituted to a degree greater than in the absence of the interaction of the test proteins.
  • the host cell may be any type of cell, including yeast, bacterial, or mammalian cell.
  • the preferred host cell is a bacterial cell.
  • the first test protein may be derived from a bacterial protein, a viral protein, an oncogene-encoded protein, a growth factor or an enzyme.
  • the hybrid proteins may be encoded on a library of plasmids containing DNA inserts that are derived from genomic DNA, cDNA, or synthetically generated DNA sequences fused to the DNA sequence encoding the second amino acid domain.
  • EXAMPLE The method and components of the kit for the present invention were tested using GAL4 as the transeriptional activator and three interacting test proteins, EGF receptor, Grb2 and Sos from the recently dissected signal transduction pathway of epidermal growth factor (EGF) stimulated mitogenesis, as reviewed by Schlessinger and Ulrich, in Neuron, 9, 383-391 (1992) .
  • the successful analysis of the pathway relies on defining components coupling the stimulated EGF receptor to Ras activation.
  • the EGF receptor protein is a receptor protein for epidermal growth factor (EGF) .
  • the Grb2 protein growth factor receptor bound protein 2
  • the SH2 domain of Grb2 binds to a number of tyrosine phosphorylated proteins, including autophosphorylated EGF receptor after EGF activation. See Lowenstein et al . , Cell, 70, 431-442 (1992) ; Buday et al .
  • the Sos protein (guanine-nucleotide exchange releasing factor Son of sevenless) is a guanine-nucleotide exchange factor for Ras proteins and the SH3 domains of Grb2 binds to proline-rich motifs such as those found in the carboxyl terminal region of Sos.
  • the EGF triggered tyrosine phosphorylation event in the EGF receptor is conveyed through Grb2 to Sos which further relays the signal by regulating the Ras activation site.
  • FIG.l is a representational map of the plasmid, pDela, for expression of a third protein- hybrid in yeast nuclei .
  • the shuttle vector plasmid contains an Amp r gene and a Col El replication sequence for ampicillin-resistance selection and propagation in E. coli ; a 2 ⁇ Ori replication sequence and a Ura3 + gene for propagation and selection in Ura " yeast strain to grow on uracil drop-out media.
  • MCS multiple cloning sites
  • NLS SV40 T antigen nuclear localization sequence
  • PADH1 with eye transcription termination sequence (T cycl) .
  • Fig.2 is a schematic representation of three DNA fragments encoding the formation of detecting formation of ternary complex of EGF receptor, Grb2 and Sos in the yeast three-hybrid system. Construction of three-hybrid expressing plasmids is described below. Fragments or whole protein (filled bars) are obtained and subcloned to make pGBT9-EGFR for expressing GAL4- BD and cytoplasmic domain of EGF receptor fusion protein. Fragments or whole protein (filled bars) are obtained and subcloned to make pDela-Grb2 for expressing SV40 T antigen nuclear localization domain and Grb2 fusion protein. Fragments or whole protein
  • TM transmembrane domain
  • TK tyrosine kinase domain
  • Gex guanine- nucleotide exchange catalytic domain
  • PP proline rich domain
  • the plasmids used in this example were obtained or constructed as follows:
  • Plasmid Construction -- pDela (5.6kb) was constructed by ligating a 1.7 kb NspB II - Kpn I fragment of pGAD424 (from nucleotide 5420 to 479) and a 3.9 kb Hpa I - Kpn I fragment of pYes2 (from nucleotide 2284 to 354, pYes2 was from Invitrogen, San Diego, CA) in an orientation such that the two Kpn I complementary ends joined together, and the blunt end of NspB II and Hpa I joined together. The multiple cloning site region and the junction region of fragments were confirmed by DNA sequencing.
  • the cytoplasmic domain (amino acid 671-1211) of the murine EGF receptor was obtained by reverse transcriptase and polymerase chain reactions (RT-PCR) using mouse brain mRNA.
  • the antisense primer including a Pst I site (underlined) has the sequence of 5'CCC CTG CAG TCA TGC TCC AAT AAA CTC ACT GC3' .
  • the sense primer including a Sma I site has a sequence of 5'T CCC CCG GGG CGA AGA CGT CAC ATT GTT CGA AA3' .
  • the amplified fragment was cloned into the Sma I/Pst I sites of pGBT9 to create pGBP9-EGFR.
  • Murine Grb2 was obtained by RT-PCR using mouse brain mRNA.
  • the antisense primer including an EcoRI site has the sequence of 5'G GAA TTC TTA GAC GTT CCG GTT CAC TGG G3' .
  • the sense primer including a site BamH I had a sequence of 5' CG GGA TCC GAA GCC ATC GCC AAA TAT GAC3' .
  • the amplified fragment was cloned into the BamH I/EcoR- I sites of pDela to create pDela-Grb2.
  • the carboxyl terminal domain (amino acid 1092- 1297) of the murine Sos2 (20) was obtained by RT-PCR using mouse brain mRNA.
  • the antisense primer including a BamH I site had the sequence of 5'GG GGA TCC TCA TTG AGG AGT TTT CTG CAT T3 ' .
  • the sense primer including a Sma I site had a sequence of 5'T CCC CCG GGG AAG ACT TTC TTC AGC TCA TGT3' .
  • the amplified fragment was cloned into the Sma 1/BamH I sites of pGAD424 to create pGAD424-Sos2. All inserts were verified by DNA sequencing.
  • BY1361 (MATa, leu2-3 , trpl -901 , his3 -200, ura3 -52, ade2 -101 , gal4 -542 , gal 80 -538, GALl-lacZ, GAL1-His3) which is deleted for both GAL4 and GAL80, a negative regulator of GAL4, and which also contains a GALl-lacZ fusion gene, as described by Gill and Ptashne, in Cell, 51, 121-126 (1987) .
  • ⁇ -galactosidase activity is a measure of GAL4 function derived from the plasmid-borne GAL4 constructs.
  • the strain also contains mutations of the HIS3, LEU2 and URA3 genes, which are the selectable genes on plasmids containing the DNA-binding domain, the activation domain, and the third hybrid protein, respectively.
  • Transformants were grown in media which can induce transcription from UAS G .
  • the media contained 2% galactose, 2% ethanol, 2% glycerol, and did not contain either leucine or histidine, or both, as appropriate in order to maintain the plasmids.
  • X-gal provides the chemical color change indicating cleavage by an intact 0-galactosidase.
  • X- gal is an abbreviation for 5-bromo-4-chloro-3-indolyl- /3-D-galactopyranoside. Accordingly, the yeast cells containing the interacting hybrid proteins which produced blue colonies are easily differentiated from a background of white cells containing only a single hybrid and not expressing .-galactosidase activity.
  • pDela contains a Ura3 + gene so that triple transformants of pDela pGBT9- and pGAD424- plasmids can be selected on a Ura " Trp " Leu " yeast background.
  • High level constitutive transcription of the third hybrid is driven by the strong promoter of house ⁇ keeping alcohol dehydrogenase gene 1 (PADH1) .
  • PADH1 alcohol dehydrogenase gene 1
  • the third hybrid protein is targeted to yeast nuclei by the SV40 T antigen nuclear localization signal.
  • EGF receptor does not contact Sos directly. But rather the phosphorylated tyrosine in EGF receptor binds to the SH2 domain in Grb2 whose SH3 domains bind Sos. Thus, a complex of EGF receptor/Grb2/Sos is formed.
  • a strain BY3161 that is Trp " Leu " Ura " and contains reporter genes LacZ and His3 + under the control of GAL4 up-stream activation sequence.
  • yeast two-hybrid system can be expanded to include a third hybrid for detecting ternary complex formation.
  • the three-hybrid system confirms previous in vi tro co-immunoprecipitation studies showing EGF receptor/Grb2/Sos2 complex formation upon EGF activation (Lowenstein et al . , Cell, 70, 431-442 (1992) ; Buday et al. , Cell, 73, 611-620 (1993) ; Li et al., Nature, 363, 85-88 (1993)) . Since yeast provides an environment closely mimicing in vivo situation, this result strongly supports the notion that in EGF stimulation cells, the signal is transduced via EGF receptor/Grb2/Sos complex to Ras activation.

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Abstract

L'invention concerne un procédé et un kit permettant de détecter des interactions entre au moins trois protéines, in vivo, par reconstitution de l'activité d'un activateur transcriptionnel. La reconstitution de l'activateur transcriptionnel se fait au moyen de gènes chimères qui expriment des protéines hybrides. Selon un mode de réalisation, trois types de protéines hybrides sont préparés. La première protéine hybride contient le domaine de liaison d'ADN d'un activateur transcriptionnel fusionné avec la première protéine test. La seconde protéine hybride contient un domaine d'activation transcriptionnel fusionné avec la seconde protéine test. La troisième protéine hybride contient un peptide de localisation nucléaire fusionné avec une troisième protéine test et induit l'assemblage du complexe à trois protéines impliquant les trois protéines hybrides. Si les trois protéines test peuvent interagir, elles mettent les deux domaines de l'activateur transcriptionnel à proximité immédiate l'un de l'autre. Cette proximité suffit pour provoquer la transcription, laquelle peut être détectée par l'activité d'un gène marqueur qui contient un site de liaison pour le domaine de liaison d'ADN.
PCT/IB1996/001495 1995-12-29 1996-12-30 Procede et kit pour la detection d'interactions entre plusieurs proteines WO1997024457A1 (fr)

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JP2005532071A (ja) * 2002-07-10 2005-10-27 ストラタジーン カリフォルニア 足場としてのヒト化レニラ・レニフォルミス緑色蛍光タンパク質
US20060099713A1 (en) * 2002-10-01 2006-05-11 Buck Institute Targeted-assisted iterative screening (tais):a novel screening format for large molecular repertoires
WO2013049398A2 (fr) 2011-09-28 2013-04-04 H. Lee Moffitt Cancer Center & Research Institute, Inc. Interaction protéine-protéine en tant que biomarqueurs

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